Method and carrier complexes for delivering molecules to cells

ABSTRACT

The invention relates to carrier complexes and methods for delivering molecules to cells. The carrier complexes comprises a molecule and an aromatic cationic peptide in accordance with the invention. In one embodiment, the method for delivering a molecule to a cell comprises contacting the cell with a carrier complex. In another embodiment, the method for delivering a molecule to a cell comprises contacting the cell with a molecule and an aromatic cationic peptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/631,048, filed Dec. 4, 2009, which is a continuation of U.S.application Ser. No. 10/838,135, filed on May 3, 2004, which claimspriority to U.S. Provisional Application Ser. No. 60/467,516, filed onMay 1, 2003. The specifications of the foregoing applications are herebyincorporated by reference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support from the NationalInstitute on Drug Abuse under Grant No. P01-DA-08924. The U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Biological cells are generally highly selective as to the molecules thatare allowed to pass through the cell membrane. As such, the delivery ofcompounds, such as small molecules and biological molecules into a cellis usually limited by the physical properties of the compound. The smallmolecules and biological molecules may, for example, be pharmaceuticallyactive compounds.

The lack of delivery of such molecules, including macromolecules, suchas proteins and nucleic acids, into cells in vivo, has been an obstacleto the therapeutic, prophylactic and/or diagnostic use of a large numberof potentially effective compounds.

In addition, many compounds which appear promising in vitro, have beendiscarded as potential drugs due to the lack of ability to deliver thecompound effectively inside a cell, in vivo.

Several reports have addressed the problem of delivering compounds tocells by covalently attaching the compounds to “protein transductiondomains” (PTDs). Schwarze et al. (Trends Pharmacol Sci. 2000; 21:45-8)and U.S. Pat. No. 6,221,355 to Dowdy disclose several PTDs that cancross the lipid bilayer of cells in a concentration-dependent manner.The PTDs disclosed include PTDs derived from the HIV-I tat protein, froma Drosophila homeotic transcription factor encoded by the antennapedia(abbreviated ANTP) gene, and from a herpes simplex virus VP22transcription factor. The HIV-1 tat PTD is eleven amino acids in length,the ANTP PTD is sixteen amino acids in length, and the VP22 PTD is 34amino acids in length.

Recent publications, however, indicate that these PTDs enter cells viaenergy-dependent endocytosis. Therefore, the “PTD-cargo” complexes arecontained within the cell's endosomal vesicles and not available to, forexample, the cytoplasm of the cell. Accordingly, the “PTD-cargo”complexes must be released from the endosomal vesicles in order to bebioactive (Richard et al., 1 Biol. Chem. 2003; 278:585-590; Drin et al.,J. Biol. Chem., 2003; 278:31192-31201). Further, there are recentreports that these PTDs are toxic to cells.

Thus, there is a need for peptides which are capable of crossing thelipid membrane of cells in an energy-independent non-endocytotic manner.In addition, in order to avoid immune responses, commonly known forlarge peptides, there is a need for smaller, peptidase-resistant,peptides. Finally, it is important that the peptide carriers be nontoxicto cells.

SUMMARY OF THE INVENTION

These needs have been met by the present invention which provides amethod for delivering a molecule to a cell. The method comprisescontacting the cell with a carrier complex, wherein the carrier complexcomprises the molecule and an aromatic cationic 20 peptide, and whereinthe aromatic cationic peptide comprises:

-   -   (a) at least one net positive charge;    -   (b) a minimum of three amino acids;    -   (c) a maximum of ten amino acids;    -   (d) a relationship between the minimum number of net positive        charges (p_(m)) and the total number of amino acid residues (r)        wherein 3p_(m) is the largest number that is less than or equal        to r+1; and    -   (e) a relationship between the minimum number of aromatic        groups (a) and the total number of net positive charges (p_(t))        wherein 3a is the largest number that is less than or equal to        p_(t)+1, except that when a is 1, p_(t) may also be 1.

In another embodiment, the invention provides a carrier complexcomprising a molecule and an aromatic cationic peptide, wherein thearomatic cationic peptide comprises:

-   -   (a) at least one net positive charge;    -   (b) a minimum of three amino acids;    -   (c) a maximum of ten amino acids;    -   (d) a relationship between the minimum number of net positive        charges (p_(m)) and the total number of amino acid residues (r)        wherein 3p_(m) is the largest number that is less than or equal        to r+1; and    -   (e) a relationship between the minimum number of aromatic        groups (a) and the total number of net positive charges (p_(t))        wherein 3a is the largest number that is less than or equal to        p_(t)+1, except that when a is 1, p_(t) may also be 1.

In yet another embodiment, the invention provides a method fordelivering a molecule to a cell. The method comprises contacting thecell with a molecule and an aromatic cationic peptide, wherein thearomatic cationic peptide comprises:

-   -   (a) at least one net positive charge;    -   (b) a minimum of three amino acids;    -   (c) a maximum of ten amino acids;    -   (d) a relationship between the minimum number of net positive        charges (p_(m)) and the total number of amino acid residues (r)        wherein 3p_(m) is the largest number that is less than or equal        to r+1; and    -   (e) a relationship between the minimum number of aromatic        groups (a) and the total number of net positive charges (p_(t))        wherein 3a is the largest number that is less than or equal to        p_(t)+1, except that when a is 1, p_(t) may also be 1.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Peptide uptake in Caco-2 cells. Time course of [³H][Dmt¹]DALDA(A) and [¹⁴C]Gly-Sar (B) uptake. Caco-2 cells were incubated with[³H][Dmt¹]DALDA (250 nM, 47 Ci/mmol) or [¹⁴C]Gly-Sar (50 μM, 56.7mCi/mmol) for 1 h at either 37 or 4° C. Radioactivity was subsequentlydetermined in solubilized cells. (C) effect of acid-wash on accumulationof [³H][Dmt¹]DALDA. Caco-2 cells were incubated with [³H][Dmt¹]DALDA for1 h at 37° C. Before cell lysis, cells were subjected to acid-wash toremove cell surface-associated radioactivity. (D) effect of [Dmt¹]DALDAconcentration on [Dmt¹]DALDA uptake. Cells were incubated with a rangeof [Dmt¹]DALDA concentrations (1 μM-3 mM) for 1 h at 37° C. All data arepresented as mean.±S.E. of three independent monolayers. Where errorbars are not apparent, they are smaller than the symbol.

FIG. 2. Effect of pH and DEPC on [³H][Dmt¹]DALDA (A and C) and[¹⁴C]Gly-Sar (B and D) uptake in Caco-2 cells. Caco-2 cells wereincubated with [³H][Dmt¹]DALDA (250 nM, 47 Ci/mmol) or [¹⁴C]Gly-Sar (50μM, 56.7 mCi/mmol) for 1 h at 37° C. under various pH conditions (A andB). Cells were preincubated at 25° C. with 0.2 mM DEPC for 10 min beforeincubation with [³H][Dmt¹]DALDA (250 nM, 47 Ci/mmol) or [¹⁴C]Gly-Sar (50μM, 56.7 mCi/mmol) at 37° C. for 1 h (C and D). All data are presentedas mean±S.E. of three independent monolayers.

FIG. 3. (A) uptake of [³H][Dmt¹]DALDA in different cell lines. Cellswere incubated with [³H][Dmt¹]DALDA (250 nM, 47 Ci/mmol) for 1 h at 37°C. Before cell lysis, cells were subjected to acid-wash to remove cellsurface-associated radioactivity. Data shown represent acid-resistantradioactivity and are presented as mean±S.E. for three independentmonolayers. (B) specific binding of [³H][Dmt¹]DALDA to cell membranes.Membranes prepared from SH-SY5Y cells and Caco-2 cells were incubatedwith [³H][Dmt¹]DALDA (15-960 μM) for 1 h at 25° C. Nonspecific bindingwas assessed by inclusion of 1 μM unlabeled [Dmt¹]DALDA. Freeradioligand was separated from bound radioligand by rapid filtration. Nospecific binding was observed with Caco-2 cells. For SH-SY5Y cells, theKd value was 118 μM (range 87-149) and the B_(max) value was 96 fmol/mgprotein.

FIG. 4. (A) efflux of [³H][Dmt¹]DALDA (filled column) and [¹⁴C]Gly-Sar(open column). Caco-2 cells were preloaded with [³H][Dmt¹]DALDA (250 nM,47 Ci/mmol) or [¹⁴C]Gly-Sar (50 μM, 56.7 mCi/mmol) for 1 h at either 37or 4° C. Cells were then washed and incubated with culture medium for 1h at either 37 or 4° C. Radioactivity was determined in both medium andcell lysate, and the data are presented as percentage of peptideeffluxed into medium. (B) effect of DEPC on [³H][Dmt¹]DALDA efflux.Cells were preincubated with 0.2 mM DEPC for 10 min at 25° C. beforeloading with [³H][Dmt¹]DALDA. (C) effect of verapamil, an inhibitor ofp-glycoprotein, on efflux (C) and uptake (D) of [³H][Dmt¹]DALDA-.

FIG. 5. Transport of [³H][Dmt¹]DALDA and [¹⁴C]Gly-Sar across a Caco-2monolayer. Caco-2 cells (2×10⁵) were seeded on microporous membraneinside Transwell cell culture chambers. Apical-to-basolateral transportof peptides was determined by adding [³H][Dmt¹]DALDA or [¹⁴C]Gly-Sar tothe apical compartment, and 20-μl aliquots were removed from both apicaland basolateral compartments at various times after peptide addition fordetermination of radioactivity.

FIG. 6. Cellular uptake of [Dmt¹,dnsDap⁴]DALDA and [Dmt¹, atnDap⁴]DALDA.Caco-2 cells were incubated with 0.1 μM [Dmt¹,dnsDap⁴]DALDA for 15 minat 37° C. Cells were then washed and covered with PBS. Microscopy wascarried out within 10 min at room temperature. Excitation was performedat 340 nm and emission was measured at 520 nm. The fluorescence appeareddiffuse throughout the cytoplasm but was completely excluded from thenucleus. The lack of vesicular concentration at 37° C. suggestsnon-endocytotic uptake.

FIG. 7. Mass spectrometric confirmation of coupling of three peptides tocross-linker SMCC. SMCC (1 μg) and peptide (5 μg) were dissolvedtogether in 2 ml of PBS, incubated at room temperature for 30 min, andstored at 4° C. An aliquot of sample was mixed with matrix (saturated3-hydroxy picolinic acid (HPA) in 50% acetonitrile, 10 mg/ml ammoniumcitrate) in a 1:10 ratio, and spotted on a stainless steel target plate.Samples were analyzed by Matrix Assisted Laser Desorption IonizationTime-of-Flight Mass Spectrometry (MALDI-TOF MS). The molecular weightsof the peptides and their respective SMCC conjugates are indicated onthe spectra.

FIG. 8. Ability of peptides to enhance uptake of β-galactosidase (β-Gal)into N₂A neuroblastoma cells. Cells (N₂A neuroblastoma cells or Caco-2)were plated in 96-well plates (2×10⁴ cells/well) and incubated with(β-Gal or β-Gal conjugated with peptide (via SMCC) for 1 h at 37° C.Cells were then washed 4 times with phosphate buffer. The cells werethen stained with β-gal staining set (Roche) for at least 2 h at 37° C.and examined under the microscope. (A) no uptake of β-Gal was observedwhen Caco-2 cells were incubated with β-Gal. (B) presence of blue cellsindicate uptake of β-Gal conjugated with [Dmt¹]DALDA in Caco-2 cells.(C) enhanced uptake of β-Gal conjugated with [D-Arg-Dmt-Lys-Phe-NH₂] inCaco-2 cells. (D) enhanced uptake of β-Gal conjugated with [Phe¹]DALDAin Caco-2 cells. Conjugation of β-Gal with SMCC alone did not enhanceuptake.

FIG. 9. Co-incubation with [Dmt¹]DALDA-SMCC conjugate enhances uptake ofgreen fluorescent protein (GFP) into Huh7 cells. Huh7 cells (1×10⁶cells/well) were washed with DMEM and then incubated with 0.5 ml DMEMcontaining 3 μg GFP alone (A), 3 μg GFP and 40 μl [Dmt¹]DALDA (B), or 3μg GFP and 40 μl [Dmt¹]DALDA conjugated to SMCC(C) for 60 min at 37° C.2 ml of cell medium was then added to cells and incubated for anadditional 24 hours in cells incubator. After incubation, cells werewashed four times in cell medium and GFP retained in living cells wasvisualized by confocal laser scanning microscopy. Excitation wasperformed at 340 nm and emission was measured at 520 nm. Top panelrepresents images of GFP through 0.8 μM thick central horizontal opticalsection of Huh7 cells. Bottom panel represents differential interfacecontrast images in same field.

FIG. 10. Conjugation of [Dmt¹]DALDA with an RNA oligo. Synthetic RNAoligo (40 nucleotides long) was phosphorylated at the 5′ end usingγ-³²P-ATP and polynucleotide kinase. The product was purified by gelelectrophoresis. 500,000 cpm of gel-purified RNA oligo was conjugatedwith [Dmt¹]DALDA in the presence of 1 mg EDC(N-[3-dimethylaminopropyl-N′-ethylcarboiimide]). The product of theconjugation reaction ([Dmt¹]DALDA-RNA oligo) and RNA oligo alone wereanalyzed on 15% polyacrylamide urea gel.

FIG. 11. Uptake of [Dmt¹]DALDA-[³²P]RNA oligo conjugate into Caco-2cells. Caco-2 cells (1×10⁶) were washed three times in DMEM medium andpreincubated in DMEM for 5 minutes. Cells were then incubated with[Dmt¹]DALDA-[³²P]RNA oligo conjugate or control RNA (approximately20,000 cpm) for 60 minutes at 37° C. After incubation, the cells werewashed, lysed, and radioactivity determined in the cell lysate. Theuptake of [Dmt¹]DALDA-[³²P]RNA conjugate was >3-fold greater compared toRNA alone.

FIG. 12. Effect of peptide-SMCC conjugates to enhance uptake of RNAoligo into Huh7 cells. (A) Effect of time on cell uptake of RNA oligo.Huh7 cells (1×10⁶ cells/well) were washed with DMEM and then incubatedwith 1.0 ml DMEM containing [³²P]RNA oligo (single strand, 11 bases;˜100,000 cpm) alone or with 40 ml [Dmt¹]DALDA-SMCC conjugate for 15 or60 min at 37° C. Cells were then washed four times in DMEM and one timein sodium acetate solution to remove nonspecific binding beforeincubated in lysis buffer for 30 min and retained radioactivitydetermined. Co-incubation of RNA oligo with [Dmt¹]DALDA-SMCC at 37° C.increased uptake of the RNA oligo by 10-fold after 15 min incubation,and 20-fold after 60 min incubation. (B) Effect of temperature on celluptake of RNA oligo. The ability of [Dmt¹]DALDA-SMCC to enhance RNAuptake was less at 4° C., although it was still increased uptake by10-fold. (C). Enhanced cellular uptake of RNA by different peptide-SMCCconjugates. Huh7 cells (1×10⁶ cells/well) were washed with DMEM and thenincubated with 1.0 ml DMEM containing [³²P]RNA oligo alone or with 40 mlpeptide-SMCC conjugate for 15 minutes at 37° C. All three peptide-SMCCconjugates increased RNA uptake.

FIG. 13. Co-incubation with [Dmt¹]DALDA-SMCC conjugate enhanced uptakeof two RNAs of different lengths. [Dmt¹]DALDA was conjugated with SMCCand confirmed by mass spectroscopy. An 11-mer RNA oligo and a 1350-merRNA were mixed with the [Dmt¹]DALDA-SMCC conjugate for 15 min at roomtemperature. Huh7 cells (1×10⁶ cells/well) were washed with DMEM andthen incubated with 1 ml DMEM containing either the RNA alone (˜100,000cpm), or the RNA mixed with the [Dmt¹]DALDA-SMCC conjugate for 60 min at37° C. and 5% CO₂. The cells were then washed four times in DMEM and onetime in sodium acetate solution to remove nonspecific binding. Thewashed cells were then incubated in lysis buffer for 30 min and retainedradioactivity counted. Compared to incubation with RNA alone,co-incubation with the [Dmt¹]DALDA-SMCC conjugate increased the uptakeof the 11-mer RNA by 22-fold, and the uptake of the 1350-mer RNA by3-fold.

FIG. 14. Conjugation of DNA oligo to [Dmt¹]DALDA. SMCC (1 μg) and[Dmt¹]DALDA (5 μg) were dissolved together in 2 ml of PBS, incubated atroom temperature for 30 min, and mixed with deprotected 3′-thiol DNAoligo at 4° C. for 24 hours. After incubation, an aliquot of sample wasmixed with matrix (saturated 3-hydroxy picolinic acid (HPA) in 50%acetonitrile, 10 mg/ml ammonium citrate) in a 1:10 ratio, and spotted ona stainless steel target plate. Samples were analyzed by MALDI-TOF MS(A). The molecular weights of 3′-thiol DNA oligo and [Dmt¹]DALDA-DNAcovalent complex were found to be 6392 and 7171, respectively. Bothconjugated and unconjugated oligos were phosphorylated at the 5′-endusing γ-³²P-ATP in the reaction with polynucleotide kinase, and theproducts of kinase reaction were analyzed on 15% polyacrylamide urea geland gel-purified for cellular uptake studies (B).

FIG. 15. Cellular uptake of DNA oligo conjugated with [Dmt¹]DALDA. A3′-thiol-modified 20-mer DNA was conjugated to [Dmt¹]DALDA using SMCC,and the formation of the conjugate was confirmed by mass spectroscopy.Both conjugated and unconjugated DNA oligos were radiolabeled at the5′-end with ³²P and gel-purified. Neuronal N₂A (1×10⁶ cells/well) cellswere washed with DMEM and incubated with 1 ml DMEM containing either[Dmt¹]DALDA-conjugated or unconjugated DNA oligo (˜100,000 cpm) for 2 hor 19 h at 37° C. and 5% CO₂. Cells were then washed four times in DMEMand one time in sodium acetate solution to remove nonspecific binding.The cells were then incubated in lysis buffer for 30 min and retainedradioactivity determined. Y-axis shows uptake of DNA represented aspercent of total radioactivity.

FIG. 16. [Dmt¹]DALDA is not toxic to cells in culture. Neuronal N₂ cellswere incubated with [Dmt¹]DALDA (1 nM to 10 μM) for 24 h and cellviability was determined by the MTT assay.

FIG. 17. [Dmt¹]DALDA-SMCC conjugate does not induce apoptosis in Huh7cells. Huh7 cells (1×10⁶ cells/well) were washed three times in DMEM,and 1 ml of fresh medium was applied. Then, either 50 μl of[Dmt¹]DALDA-SMCC conjugate (1 mM) in PBS or PBS only (control) was addedto the cell medium and incubated at 37° C. for 24 hours at 5% CO₂. Afterincubation, 1 μl of Hoechst dye for staining apoptotic nuclei was addedto the cells and incubated for an additional 15 min. Excessive Hoechstdye was removed by washing cells with cell medium (free of pH indicator)and cells treated with [Dmt¹]DALDA-SMCC conjugate were compared withcontrol cells using fluorescent microscopy (excitation at 350 nm andemission at 461 nm).

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the surprising discovery by the inventors thatcertain carrier complexes comprising at least one molecule and anaromatic cationic peptide can cross cell membranes by anenergy-independent mechanism and deliver the molecules inside the cell.

Aromatic Cationic Peptides

The aromatic cationic peptides useful in the present invention have anet positive charge as described below, are water-soluble and highlypolar. The peptides include a minimum of three amino acids, andpreferably include a minimum of four amino acids, covalently joined bypeptide bonds.

The maximum number of amino acids present in the aromatic cationicpeptides is ten, preferably about eight, and most preferably about six.Optimally, the number of amino acids present in the peptides is aboutfour. The term “about” as used in the definition for the maximum numberof amino acids means plus or minus one amino acid.

The amino acids of the aromatic cationic peptides useful in the presentinvention can be any amino acid. As used herein, the term “amino acid”is used to refer to any organic molecule that contains at least oneamino group and at least one carboxyl group. Preferably, at least oneamino group is at the a position relative to the carboxyl group.

The amino acids may be naturally occurring. Naturally occurring aminoacids include, for example, the twenty most common amino acids normallyfound in proteins, i.e., alanine (Ala), arginine (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Glu), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ileu), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),and valine (Val).

Other naturally occurring amino acids include, for example, amino acidsthat are synthesized in metabolic processes not associated with proteinsynthesis. For example, the amino acid ornithine is synthesized inmammalian metabolism during the production of urea.

The aromatic cationic peptides useful in the present inventionoptionally comprise one or more amino acids that are non-naturallyoccurring. In one embodiment, the peptide has no amino acids that arenaturally occurring.

Non-naturally occurring amino acids are those amino acids that typicallyare not synthesized in normal metabolic processes in living organisms,and do not naturally occur in proteins.

In addition, the non-naturally occurring amino acids useful in thepresent invention preferably are also not recognized by commonproteases. Thus, the non-naturally occurring amino acids are preferablyresistant, and more preferably insensitive, to common proteases.

Non-naturally occurring amino acids can be present at any position inthe peptide. For example, a non-naturally occurring amino acid can be atthe N-terminus, the C-terminus, and/or at any one or more positionsbetween the N-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups. Some examples of alkyl amino acids includeα-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid,.delta.-aminovaleric acid, and .epsilon.-aminocaproic acid. Someexamples of aryl amino acids include ortho-, meta, and para-aminobenzoicacid. Some examples of alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives ofnaturally occurring amino acids. The derivatives of naturally occurringamino acids may, for example, include the addition of one or morechemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkoxy(i.e., alkoxy), amino, C₁-C₄ alkylamino (e.g., methylamino) and C₁-C₄dialkylamino (e.g., dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva), norleucine (Nle), and hydroxyproline (Hyp).

Another example of a modification of an amino acid in a peptide usefulin the present invention is the derivatization of a carboxyl group of anaspartic acid or a glutamic acid residue of the peptide. One example ofderivatization is amidation with ammonia or with a primary or secondaryamine, e.g., methylamine, ethylamine, dimethylamine or diethylamine.Another example of derivatization includes esterification with, forexample, methyl or ethyl alcohol.

Another such modification includes modification of an amino group of alysine, arginine, or histidine residue. For example, such amino groupscan be acylated. Some suitable acyl groups include, for example, abenzoyl group or an alkanoyl group comprising any of the C₁-C₄ alkylgroups mentioned above, such as an acetyl or propionyl group.

The non-naturally occurring amino acids may generally be levorotatory(L-), dextrorotatory (D), or mixtures thereof. Examples of suitablenon-naturally occurring amino acids also include the dextrorotatory (D-)form of any of the above-mentioned naturally occurring L-amino acids, aswell as L- and/or D-non-naturally occurring amino acids. In this regard,it should be noted that D-amino acids do not normally occur in proteins,although they are found in certain peptide antibiotics that aresynthesized by means other than the normal ribosomal protein syntheticmachinery of the cell. As used herein, such D-amino acids are consideredto be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides useful in theinvention should have less than five, preferably less than four, morepreferably less than three, and most preferably, less than twocontiguous L-amino acids recognized by common proteases, irrespective ofwhether the amino acids are naturally or non-naturally occurring. In oneembodiment, the peptide has only D-amino acids, and no L-amino acids.

If the peptide contains protease sensitive sequences of amino acids, atleast one of the amino acids is preferably a non-naturally-occurringD-amino acid, thereby conferring protease resistance. An example of aprotease sensitive sequence includes two or more contiguous basic aminoacids that are cleaved by common proteases, such as endopeptidases andtrypsin. Examples of basic amino acids include arginine, lysine andhistidine.

It is important that the aromatic cationic peptides have a minimumnumber of net positive charges at physiological pH in comparison to thetotal number of amino acid residues in the peptide. The minimum numberof net positive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r).

The minimum number of net positive charges discussed below are all atphysiological pH. The term “physiological pH” as used herein refers tothe normal pH in the cells of the tissues and organs of the mammalianbody. For instance, the physiological pH of a human is normallyapproximately 7.4, but normal physiological pH in mammals may be any pHfrom about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-Arg (SEQ ID NO: 1) has onenegatively charged amino acid (i.e., Glu) and four positively chargedamino acids (i.e., two Arg residues, one Lys, and one His). Therefore,the above peptide has a net positive charge of three.

In one embodiment of the present invention, the aromatic cationicpeptides have a relationship between the minimum number of net positivecharges at physiological pH (p_(m)) and the total number of amino acidresidues (r) wherein 3p_(m) is the largest number that is less than orequal to r+1. In this embodiment, the relationship between the minimumnumber of net positive charges (p_(m)) and the total number of aminoacid residues (r) is as follows:

(r) 3 4 5 6 7 8 9 10 (p_(m)) 1 1 2 2 2 3 3 3

In another embodiment, the aromatic cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

(r) 3 4 5 6 7 8 9 10 (p_(m)) 2 2 3 3 4 4 5 5

In one embodiment, the number of net positive charges (p_(m)) and thenumber of amino acid residues (r) are equal. In another preferredembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, preferably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a).

Naturally occurring amino acids that have an aromatic group include theamino acids histidine, tryptophan, tyrosine, and phenylalanine. Forexample, the hexapeptide Lys-Gln-Tyr-Arg-Phe-Trp (SEQ ID NO: 2) has anet positive charge of two (contributed by the lysinc and arginineresidues) and three aromatic groups (contributed by tyrosine,phenylalanine and tryptophan residues).

In one embodiment of the present invention, the aromatic cationicpeptides useful in the methods of the present invention have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges at physiological pH (p_(t)) wherein3a is the largest number that is less than or equal to p_(t)+1, exceptthat when p_(t) is 1, a may also be 1. In this embodiment, therelationship between the minimum number of aromatic groups (a) and thenumber of net positive charges (p_(t)) is as follows:

(p_(t)) 1 2 3 4 5 6 7 8 9 10 (a) 1 1 1 1 2 2 2 3 3 3

In another embodiment the aromatic cationic peptides have a relationshipbetween the minimum number of aromatic groups (a) and the total numberof net positive charges (p_(t)) wherein 2a is the largest number that isless than or equal to p_(t)+1. In this embodiment, the relationshipbetween the minimum number of aromatic amino acid residues (a) and thetotal number of net positive charges (p_(t)) is as follows:

(p_(t)) 1 2 3 4 5 6 7 8 9 10 (a) 1 1 2 2 3 3 4 4 5 5

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are preferably amidated with, for example, ammonia to form aC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group.

In addition, the free carboxylate groups of amino acid residues havingmore than one carboxylate group, e.g., asparagine, glutamine, asparticacid, and glutamic acid residues, may also be amidated wherever theyoccur. The amidation at these positions may be with ammonia or any ofthe primary or secondary amines described above.

In one embodiment, the aromatic cationic peptide useful in the methodsof the present invention is a tripeptide having two net positive chargesand at least one aromatic amino acid. In a particular embodiment, thearomatic cationic peptide useful in the methods of the present inventionis a tripeptide having two net positive charges and two aromatic aminoacids.

Aromatic cationic peptides useful in the methods of the presentinvention include, but are not limited to, the following peptideexamples:

Lys-D-Arg-Tyr-NH₂, Phe-D-Arg-His, D-Tyr-Trp-Lys-NH₂,Trp-D-Lys-Tyr-Arg-NH₂, Tyr-His-D-Gly-Met, Phe-Arg-D-His-Asp,(SEQ ID NO: 3) Tyr-D-Arg-Phe-Lys-Glu-NH₂, (SEQ ID NO: 4)Met-Tyr-D-Lys-Phe-Arg, (SEQ ID NO: 5) D-His-Glu-Lys-Tyr-D-Phe-Arg,(SEQ ID NO: 6) Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂, (SEQ ID NO: 7)Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His, (SEQ ID NO: 8)Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂, (SEQ ID NO: 9)Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂, (SEQ ID NO: 10)Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys, (SEQ ID NO: 11)Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂, (SEQ ID NO: 12)Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys, (SEQ ID NO: 13)Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂, (SEQ ID NO: 14)D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg- Trp-NH₂, and(SEQ ID NO: 15) Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe.

In a particularly preferred embodiment, an aromatic cationic peptide hasthe formula Tyr-D-Arg-Phe-Lys-NH₂ (for convenience represented by theacronym: DALDA). DALDA has a net positive charge of three, contributedby the amino acids tyrosine, arginine, and lysine and has two aromaticgroups contributed by the amino acids phenylalanine and tyrosine. Thetyrosine of DALDA can be a modified derivative of tyrosine such as in2′,6′-dimethyltyrosine to produce the compound having the formula2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ (i.e., Dmt¹-DALDA). Other modifiedderivatives of tyrosine include 2′-methyltyrosine (Mmt);N,2′,6′-trimethyltyrosine (Tmt); and 2′-hydroxy-6′-methyltryosine (Hmt).

In another preferred embodiment, the amino acid at the N-terminus ofDALDA can be a phenylalanine or its derivative. An aromatic cationicpeptide with phenylalanine at the N-terminus has the formulaPhe-D-Arg-Phe-Lys-NH₂ (i.e., Phe¹-DALDA). Preferred derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp),2′,6′-dimethylphenylalanine (Dmp), N,2′,6′-trimethylphenylalanine (Tmp),and 2′-hydroxy-6′-methylphenylalanine (Hmp).

In another embodiment, the amino acid sequence of Dmt¹-DALDA isrearranged such that Dmt is not at the N-terminus. An example of such anaromatic cationic peptide has the formula D-Arg-2′6′Dmt-Lys-Phe-NH₂.

Any of the specific peptides mentioned herein, such as those mentionedabove and those mentioned below, e.g., in table 1, including Dmt¹-DALDA,DALDA, Phe¹-DALDA, D-Arg-2′,6′Dmt-Lys-Phe-NH₂ and their derivatives canfurther include functional analogs. A peptide is considered a functionalanalog of Dmt¹-DALDA, DALDA, Phe¹-DALDA, or D-Arg-2′6′Dmt-Lys-Phe-NH₂ ifthe analog has the same function as Dmt¹-DALDA, DALDA, Phe¹-DALDA, orD-Arg-2′6′Dmt-Lys-Phe-NH₂. The analog may, for example, be asubstitution variant of Dmt¹-DALDA, DALDA, Phe¹-DALDA, orD-Arg-2′6′Dmt-Lys-Phe-NH₂, wherein one or more amino acids issubstituted by another amino acid.

Suitable substitution variants of Dmt¹-DALDA, DALDA, Phe¹-DALDA, orD-Arg-2′6′Dmt-Lys-Phe-NH₂ include conservative amino acid substitutions.Amino acids may be grouped according to their physicochemicalcharacteristics as follows:

-   -   (a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);    -   (b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(O);    -   (c) Basic amino acids: His(H) Arg(R) Lys(K);    -   (d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and    -   (e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution. Conservativesubstitutions tend to preserve the physicochemical characteristics ofthe original peptide. In contrast, substitutions of an amino acid in apeptide by another amino acid in a different group is generally morelikely to alter the characteristics of the original peptide.

Examples of analogs useful in the practice of the present inventioninclude, but are not limited to the aromatic cationic peptides shown inTables 1 and 2.

TABLE 1 Amino Amino Amino Acid Acid Acid Amino Amino Posi- Posi- Posi-Acid Acid C-Terminal tion 1 tion 2 tion 3 Position 4 Position 5Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ Tyr D-Arg PheDab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂ 2′6′Dmt D-ArgPhe Lys Cys NH₂ (SEQ ID NO: 16) 2′6′Dmt D-Arg Phe Lys- NH₂ NH(CH2)2—NH-dns 2′6′Dmt D-Arg Phe Lys- NH₂ NH(CH2)2—NH- atn 2′6′Dmt D-Arg Phe dnsLysNH₂ 2′6′Dmt D-Cit Phe Lys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-ArgPhe Orn NH₂ 2′6′Dmt D-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′DmtD-Arg Phe Ahp(2- NH₂ aminoheptanoic Bio- D-Arg Phe Lys NH₂ 2′6′Dmt3′5′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg PheDab NH₂ 3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg TyrOrn NH₂ Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg TyrLys NH₂ 2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′DmtD-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′DmtOrn NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Pap NH₂3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′DmtD-Arg 3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe DapNH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂2′6′Dmt D-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys ′PheArg NH₂ 2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′DmtD-Lys Phe Dab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂Tyr D-Lys Tyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ TyrD-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂2′6′Dmt D-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys2′6′Dmt Lys NH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt DabNH₂ 2′6′Dmt D-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′DmtD-Arg Phe atnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′DmtOrn NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dap Phe Arg Tyr NH₂2′6′Dmt D-Arg Phe Arg 2′6′Dmt NH₂ 2′6′Dmt D-Lys Phe Arg 2′6′Dmt NH₂2′6′Dmt D-Orn Phe Arg 2′6′Dmt NH₂ 2′6′Dmt D-Dab Phe Arg 2′6′Dmt NH₂3′5′Dmt D-Dap Phe Arg 3′5′Dmt NH₂ 3′5′Dmt D-Arg Phe Arg 3′5′Dmt NH₂3′5′Dmt D-Lys Phe Arg 3′5′Dmt NH₂ 3′5′Dmt D-Orn Phe Arg 3′5′Dmt NH₂ TyrD-Lys Tyr Arg Tyr NH₂ Tyr D-Orn Tyr Arg Tyr NH₂ Tyr D-Dab Tyr Arg TyrNH₂ Tyr D-Dap Tyr Arg Tyr NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg 2′6′Dmt NH₂2′6′Drnt D-Lys 2′6′Dmt Arg 2′6′Drnt NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg2′6′Dmt NH₂ 2′6′Dmt D-Dab 2′6′Dmt Arg 2′6′Dmt NH₂ 3′5′Dmt D-Dap 3′5′DmtArg 3′5′Dmt NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg 3′5′Dmt NH₂ 3′5′Dmt D-Lys3′5′Dmt Arg 3′5′Dmt NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg 3′5′Dmt NH₂ Mmt D-ArgPhe Lys Mmt NH₂ Mmt D-Arg Phe Orn Mmt NH₂ Mmt D-Arg Phe Dab Mmt NH₂ MmtD-Arg Phe Dap Mmt NH₂ Tmt D-Arg Phe Lys Tmt NH₂ Tmt D-Arg Phe Orn TmtNH₂ Tmt D-Arg Phe Dab Tmt NH₂ Tmt D-Arg Phe Dap Tmt NH₂ Hmt D-Arg PheLys Hmt NH₂ Hmt D-Arg Phe Orn Hmt NH₂ Hmt D-Arg Phe Dab Hmt NH₂ HmtD-Arg Phe Dap Hmt NH₂ Mmt D-Lys Phe Lys Mmt NH₂ Mmt D-Lys Phe Orn MmtNH₂ Mmt D-Lys Phe Dab Mmt NH₂ Mmt D-Lys Phe Dap Mmt NH₂ Mmt D-Lys PheArg Mmt NH₂ Tmt D-Lys Phe Lys Tmt NH₂ Tmt D-Lys Phe Om Tmt NH₂ Tmt D-LysPhe Dab Tmt NH₂ Tmt D-Lys Phe Dap Tmt NH₂ Tmt D-Lys Phe Arg Tmt NH₂ HmtD-Lys Phe Lys Hmt NH₂ Hmt D-Lys Phe Orn Hmt NH₂ Hmt D-Lys Phe Dab HmtNH₂ Hmt D-Lys Phe Dap Hmt NH₂ Hmt D-Lys Phe Arg Hmt NH₂ Mmt D-Lys PheArg Mmt NH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab Phe Arg NH₂ Mmt D-Dap PheArg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe Arg NH₂ Tmt D-Orn Phe ArgNH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂ Tmt D-Arg Phe Arg NH₂Hmt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ Hmt D-Dab Phe Arg NH₂ HmtD-Dap Phe Arg NH₂ Hmt D-Arg Phe Arg NH₂ Dab = diaminobutyric Dap =diaminopropionic acid Drat = dimethyltyrosine Mmt = 2′-methyltyrosineTmt = N,2′,6′-trimethyltyrosine Hmt = T-hydroxy,6-methyltyrosine dnsDap= (3-dansyl-L-a,p-diaminopropionic acid atnDap =P-anthraniloyl-L-a,o-diaminopropionic acid Bio = biotin

TABLE 2 Amino Acid Amino Acid Amino Acid Amino Acid C-Terminal Position1 Position 2 Position 3 Position 4 Modification D-Arg Dmt Lys Phe NH₂D-Arg Dmt Phe Lys NH₂ D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-ArgLys Drnt Phe NH₂ D-Arg Lys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe LysD-Arg Dmt NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe D-Arg PheLys NH₂ Phe Dmt D-Arg Lys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg DmtNH₂ Lys Phe Dmt D-Arg NH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂Lys D-Arg Phe Dmt NH₂ Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂D-Arg Dmt D-Arg Dmt NH₂ D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂Trp D-Arg Phe Lys NH₂ Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ TrpD-Arg Dmt Lys NH₂ D-Arg Trp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg TrpLys Dmt NH₂ D-Arg Trp Dmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys TrpDmt NH₂ Cha D-Arg Phe Lys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl

The amino acids of the peptides shown in Tables 1 and 2 may be in eitherthe L- or the D-configuration.

Further cationic peptides can be found in U.S. Provisional ApplicationNo. 60/444,777 filed Feb. 4, 2003, which is hereby incorporated byreference.

Molecules

The molecule can be a biological molecule or a small molecule.Preferably, the biological molecule or small molecule is apharmaceutically active molecule. A pharmaceutically active molecule asused herein, is any molecule which exerts a beneficial effect in vivo.

A biological molecule is any molecule which contains a nucleic acid oramino acid sequence and has a molecular weight greater than 450. Suchnucleic acid and amino acid sequences are referred to herein as“polynucleotides” and “polyamino acids,” respectively.

Biological molecules include polynucleotides, peptide nucleic acids, andpolyamino acids, such as peptides, polypeptides, and proteins. Examplesof biological molecules which are pharmaceutically active includeendogenous peptides (e.g., vasopressin, glutathione), proteins (e.g.,interferons), hormones (e.g., human growth hormone), enzymes (e.g.,α-galactosidase), antibodies (e.g., antibody against beta-amyloid, whichcan be used to treat Alzheimers disease), neurotrophic growth factors(e.g., nerve growth factor NGF, brain-derived neutrophic factor BDNF),cytokines (e.g., platelet-derived growth factor PDGF, vascularendothelial cell growth factor VEGF), and oligonucleotides.

The oligonucleotides may comprise any sequence of nucleotides, such asDNA or RNA. The DNA and RNA sequences can be single or double-stranded.For example, DNA encoding a protein that is beneficial in assistingsurvival of a cell during stress can be conjugated to the peptides ofthe invention. Examples of such proteins include the heat shock proteins(e.g., hsp60, hsp70, etc.).

Examples of single-stranded RNA molecules include ribozymes, RNA decoys,external guide sequences for ribozymes, antisense RNAs and mRNAs. For areview of these single-stranded RNA molecules, see Sullenger et al.(Nature 2002, 418: 252-247). The description of these single-strandedRNA molecules, and the description of illnesses and diseases which canbe treated with ribozymes, RNA decoys, external guide sequences forribozymes, antisense RNAs and mRNAs molecules disclosed in Sullenger arehereby incorporated by reference.

An example of double stranded RNA is an RNA interfering molecule (i.e.,RNAi such as, for example, siRNA (i.e., small interfering RNA)). ThesiRNA can be any known to those in the art.

The siRNA can be, for instance, sufficiently complementary to a mRNA toinhibit translation of a protein implicated in a disease, condition orillness. Examples of such proteins include, for instance, β-amyloidwhich is implicated in Alzheimer's disease and the protein ras which isimplicated in cancer.

Alternatively, the siRNA can be, for example, sufficiently complementaryto an RNA produced by a virus. The RNA produced by the virus can be anyRNA which is generally required for infection of a host cell, survivalof the virus, and/or propagation of the virus. Examples of such RNAinclude Internal Ribosome Entry Site, RNA-dependent polymeraseinitiation sites, and RNA encoding viral envelope proteins, viralnucleases, and viral proteases.

Examples of viruses include, for example, hepatitis virus, such ashepatitis A, B, and C, human immunodeficiency virus, Epstein-bar virus,cytomegalovirus, and human papilloma virus.

siRNA which target virus RNAs are known to those in the art. Forexample, siRNAs which target hepatitis C virus RNAs are known to thosein the art, see Randall et al. PNAS, 2003, 100: 235-240.

The molecule can be a small molecule. Small molecules include organiccompounds, organometallic compounds, salts of organic and organometalliccompounds, monosaccharides, amino acids, and nucleotides. Smallmolecules can further include molecules that would otherwise beconsidered biological molecules, except their molecular weight is notgreater than 450. Thus, small molecules may be lipids, oligosaccharides,oligopeptides, and oligonucleotides, and their derivatives, having amolecular weight of 450 or less.

It is emphasized that small molecules can have any molecular weight.They are merely called small molecules because they do not qualify asbiological molecules, and typically have molecular weights less than450. Small molecules include compounds that are found in nature as wellas synthetic compounds. Examples of small molecules which arepharmaceutically active include antibiotics (e.g., tetracycline,penicillin, erythromycin), cytotoxic agents (e.g., doxorubicin,adriamycin), and antioxidants (e.g., vitamin E, vitamin C, betacarotene).

Carrier Complexes

At least one molecule as described above, and at least one aromaticcationic peptide as described above, associate to form a carriercomplex. The molecule and aromatic cationic peptide can associate by anymethod known to those in the art. Suitable types of associations includechemical bonds and physical bonds. Chemical bonds include, for example,covalent bonds and coordinate bonds. Physical bonds include, forinstance, hydrogen bonds, dipolar interactions, van der Waal forces,electrostatic interactions, hydrophobic interactions and aromaticstacking.

The type of association between the molecules and aromatic cationicpeptides typically depends on, for example, functional groups availableon the molecule and functional groups available on the aromatic cationicpeptide.

For a chemical bond or physical bond, a functional group on the moleculetypically associates with a functional group on the aromatic cationicpeptide. Alternatively, a functional group on the aromatic cationicpeptide associates with a functional group on the molecule.

The functional groups on the molecule and aromatic cationic peptide canassociate directly. For example, a functional group (e.g., a sulfhydrylgroup) on a molecule can associate with a functional group (e.g.,sulfhydryl group) on an aromatic cationic peptide to form a disulfide.

Alternatively, the functional groups can associate through across-linking agent (i.e., linker). Some examples of cross-linkingagents are described below. The cross-linker can be attached to eitherthe molecule or the aromatic cationic peptide.

The linker may and may not affect the number of net charges of thearomatic cationic peptide. Typically, the linker will not contribute tothe net charge of the aromatic cationic peptide. Each amino group, ifany, present in the linker will contribute to the net positive charge ofthe aromatic cationic peptide. Each carboxyl group, if any, present inthe linker will contribute to the net negative charge of the aromaticcationic peptide.

The number of molecules or aromatic cationic peptides in the carriercomplex is limited by the capacity of the peptide to accommodatemultiple molecules or the capacity of the molecule to accommodatemultiple peptides. For example, steric hindrance may hinder the capacityof the peptide to accommodate especially large molecules. Alternatively,steric hinderance may hinder the capacity of the molecule to accommodatea relatively large (e.g., seven, eight, nine or ten amino acids inlength) aromatic cationic peptide.

The number of molecules or aromatic cationic peptides in the carriercomplex is also limited by the number of functional groups present onthe other. For example, the maximum number of molecules associated witha peptide depends on the number of functional groups present on thepeptide. Alternatively, the maximum number of peptides associated with amolecule depends on the number of functional groups present on themolecule.

In one embodiment, the carrier complex comprises at least one molecule,and preferably at least two molecules, associated with anaromatic-cationic peptide. A relatively large peptide (e.g., eight, tenamino acids in length) containing several (e.g., 3, 4, 5 or more)functional groups can be associated with several (e.g., 3, 4, 5 or more)molecules.

In another embodiment, the carrier complex comprises at least onearomatic-cationic peptide, and preferably at least two aromatic cationicpeptides, associated with a molecule. For example, a molecule containingseveral functional groups (e.g., 3, 4, 5 or more) can be associated withseveral (e.g., 3, 4, or 5 or more) peptides.

In yet another embodiment, the carrier complex comprises onearomatic-cationic peptide associated to one molecule.

In one embodiment, a carrier complex comprises at least one moleculechemically bonded (e.g., conjugated) to at least one aromatic cationicpeptide. The molecule can be chemically bonded to an aromatic cationicpeptide by any method known to those in the art. For example, afunctional group on the molecule may be directly attached to afunctional group on the aromatic cationic peptide. Some examples ofsuitable functional groups include, for example, amino, carboxyl,sulfhydryl, maleimide, isocyanate, isothiocyanate and hydroxyl.

The molecule may also be chemically bonded to the aromatic cationicpeptide by means of cross-linking agents, such as dialdehydes,carbodiimides, dimaleimides, and the like. Cross-linking agents can, forexample, be obtained from Pierce Biotechnology, Inc., Rockford, Ill. ThePierce Biotechnology, Inc. web-site can provide assistance. Additionalcross-linking agent include the platinum cross-linking agents describedin U.S. Pat. Nos. 5,580,990; 5,985,566; and 6,133,038 of KreatechBiotechnology, B.V., Amsterdam, The Netherlands.

The functional group on the molecule may be different from thefunctional group on the peptide. For example, if a sulfhydryl group ispresent on the molecule, such as in β-galactosidasc or in 5′- and/or3′-end thiol modified DNA and RNA oligonucleotides, the molecule can becross-linked to the peptide, e.g., [Dmt¹]DALDA, through the 4-aminogroup of lysine by using the cross-linking reagent SMCC (i.e.,succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) from PierceBiotechnology (see Example 10 below). In another example, the 4-aminogroup of lysine of DALDA can be conjugated directly to an α-phosphategroup at the 5′-end of an RNA or DNA oligonucleotide by using thecrosslinking reagent EDC (i.e.,(N-[3-dimethylaminopropyl-N′-ethylcarboiimide]) from PierceBiotechnology (see Example 13 below).

Alternatively, the functional group on the molecule and peptide can bethe same. Homobifunctional cross-linkers are typically used tocross-link identical functional groups. Examples of homobifunctionalcross-linkers include EGS (i.e., ethylene glycolbis[succinimidylsuccinate]), DSS (i.e., disuccinimidyl suberate), DMA(i.e., dimethyl adipimidate.2 HCl), DTSSP (i.e.,3,3′-dithiobis[sulfosuccinimidylpropionate])), DPDPB (i.e.,1,4-di-[3′-(2′-pyridyldithio)-propionamido]butane), and BMH (i.e.,bis-maleimidohexane). Such homobifunctional cross-linkers are alsoavailable from Pierce Biotechnology, Inc.

To chemically bond the molecules and the peptides, the molecules,peptides, and cross-linker are typically mixed together. The order ofaddition of the molecules, peptides, and cross-linker is not important.For example, the peptide can be mixed with the cross-linker, followed byaddition of the molecule. Alternatively, the molecule can be mixed withthe cross-linker, followed by addition of the peptide. Optimally, themolecules and the peptides arc mixed, followed by addition of thecross-linker.

The chemically bonded carrier complexes deliver the molecules to a cell.In some instances, the molecule functions in the cell without beingcleaved from the aromatic cationic peptide. For example, if the aromaticcationic peptide does not block the catalytic site of the molecule, thencleavage of the molecule from the aromatic cationic peptide is notnecessary (see Example 11 below).

In other instances, it may be beneficial to cleave the molecule from thearomatic cationic peptide. The web-site of Pierce Biotechnology, Inc.described above can also provide assistance to one skilled in the art inchoosing suitable cross-linkers which can be cleaved by, for example,enzymes in the cell. Thus the molecule can be separated from thearomatic cationic peptide. Examples of cleavable linkers include SMPT(i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene),Sulfo-LC-SPDP (i.e., sulfosuccinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e.,succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate),Sulfo-LC-SPDP (i.e., sulfosuccinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e.,N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP(i.e., 3-[(2-aminoethyl)dithio]propionic acid.HCl).

In another embodiment, a carrier complex comprises at least one moleculephysically bonded with at least one aromatic cationic peptide. Anymethod known to those in the art can be employed to physically bond themolecules with the aromatic cationic peptides.

For example, the aromatic cationic peptides and molecules can be mixedtogether by any method known to those in the art. The order of mixing isnot important. For instance, molecules can be physically mixed withmodified or unmodified aromatic cationic peptides by any method known tothose in the art. Alternatively, the modified or unmodified aromaticcationic peptides can be physically mixed with the molecules by anymethod known to those in the art.

For example, the aromatic-cationic peptides and molecules can be placedin a container and agitated, by for example, shaking the container, tomix the aromatic-cationic peptides and molecules.

The aromatic cationic peptides can be modified by any method known tothose in the art. For instance, the aromatic cationic peptide may bemodified by means of cross-linking agents or functional groups, asdescribed above. The linker may and may not affect the number of netcharges of the aromatic cationic peptide. Typically, the linker will notcontribute to the net charge of the aromatic cationic peptide. Eachamino group, if any, present in the linker contributes to the netpositive charge of the aromatic cationic peptide. Each carboxyl group,if any, present in the linker contributes to the net negative charge ofthe aromatic cationic peptide.

For example, [Dmt¹]DALDA can be modified, through the 4-amino group oflysine by using the cross-linking reagent SMCC (i.e., succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) from PierceBiotechnology (sec Example 10 below). To form a carrier complex, themodified aromatic-cationic peptide is usually formed first and thenmixed with the molecule.

One advantage of the physically bonded carrier complexes, is that themolecule functions in a cell without the need for removing an aromaticcationic peptide, such as those carrier complexes in which the moleculeis chemically bonded to an aromatic cationic peptide. Furthermore, ifthe aromatic cationic peptide does not block the catalytic site of themolecule, then dissociation of the complex is also not necessary (seeExample 12 below).

Synthesis of the Aromatic Cationic Peptides

The peptides useful in the methods of the present invention may bechemically synthesized by any of the methods well known in the art.Suitable methods for synthesizing the protein include, for example,those described by Stuart and Young in “Solid Phase Peptide Synthesis,”Second Edition, Pierce Chemical Company (1984), and in “Solid PhasePeptide Synthesis,” Methods Enzymol. 289, Academic Press, Inc, New York(1997).

Modes of Administration

In one embodiment, the invention relates to a method for delivering amolecule to a cell. The method comprises contacting a cell with amolecule and an aromatic cationic peptide. The cell can be contactedwith the molecule and aromatic cationic peptide by any method known tothose in the art. For example, a cell can be incubated with the moleculeand aromatic cationic peptide in vitro. In one aspect, the molecule andaromatic cationic peptide can be present in the form of a carriercomplex, such as those carrier complexes described above, comprisingchemically bonded or physically bonded molecules and aromatic cationicpeptides.

In another embodiment, the method for delivering a molecule to a cellcomprises contacting the cell with a carrier complex. The molecule isdelivered to the cell by contacting the cell with the carrier complexcomprising the molecule and an aromatic cationic peptide. The cell canbe contacted with the carrier complex by any method known to those inthe art.

For example, a cell can be incubated with the carrier complex in vitro.The cell can be any cell. The cell can be of plant, animal, or bacterialorigin. An example of a plant cell includes Arabidopsis cells. Examplesof bacterial cells include Saccharomyces and Lactobacillus. Animal cellsinclude mammalian cells, such as neuronal cells, renal epithelial cells,kidney cells, vascular endothelial cells, glial cells, intestinalepithelial cells and hepatocytes. An example of a vascular endothelialcell is a blood brain barrier endothelial cell.

Alternatively, the carrier complex can be administered to a mammal invivo. An effective amount of a carrier complex, preferably in apharmaceutical composition, may be administered to a mammal in needthereof by any of a number of well-known methods for administeringpharmaceutical compounds.

The carrier complex may be administered systemically or locally. In oneembodiment, the carrier complex is administered intravenously. Forexample, the carrier complex may be administered via rapid intravenousbolus injection. Preferably, however, the carrier complex isadministered as a constant rate intravenous infusion.

The carrier complexes may be administered to the tissues of a mammallocally, e.g., by injection into tissues which are accessible by asyringe. For example, if the carrier complex contains a cytotoxic agentwhich is to be delivered to a tumor in a mammal, preferably, the tumoris accessible to local administration. Such tumors include, for example,skin cancer and breast cancer.

The carrier complex may also be administered orally, topically,intranasally, intramuscularly, subcutaneously, or transdermally. In apreferred embodiment, transdermal administration of carrier complex isby iontophoresis, in which the carrier complex is delivered across theskin by an electric current.

Other routes of administration include intracerebroventricularly orintrathecally. Intracerebroventiculatly refers to administration intothe ventricular system of the brain. Intrathecally refers toadministration into the space under the arachnoid membrane of the brainor spinal cord. Thus intracerebroventricular or intrathecaladministration may be preferred for those diseases and conditions whichaffect the organs or tissues of the central nervous system.

The carrier complex useful in the methods of the invention may beadministered to mammals by sustained release, as is known in the art.Sustained release administration is a method of drug delivery to achievea certain level of the drug over a particular period of time. The leveltypically is measured by serum concentration.

Any formulation known in the art of pharmacy is suitable foradministration of the carrier complex. For oral administration, liquidor solid formulations may be used. Some examples of formulations includetablets, gelatin capsules, pills, troches, elixirs, suspensions, syrups,wafers, chewing gum and the like. The peptides can be mixed with asuitable pharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art. Examples of carriers and excipients includestarch, milk, sugar, certain types of clay, gelatin, lactic acid,stearic acid or salts thereof, including magnesium or calcium stearate,talc, vegetable fats or oils, gums and glycols.

For systemic, intracerebroventricular, intrathecal, topical, intranasal,subcutaneous, or transdermal administration, formulations of the carriercomplex may utilize conventional diluents, carriers, or excipients etc.,such as are known in the art can be employed to deliver the carriercomplex. For example, the formulations may comprise one or more of thefollowing: a stabilizer, a surfactant, preferably a nonionic surfactant,and optionally a salt and/or a buffering agent. The carrier complex maybe delivered in the form of an aqueous solution, or in a lyophilizedform.

The stabilizer may, for example, be an amino acid, such as for instance,glycine; or an oligosaccharidc, such as for example, sucrose, tetralose,lactose or a dextran. Alternatively, the stabilizer may be a sugaralcohol, such as for instance, mannitol; or a combination thereof.Preferably the stabilizer or combination of stabilizers constitutes fromabout 0.1% to about 10% weight for weight of the carrier complex.

The surfactant is preferably a nonionic surfactant, such as apolysorbate. Some examples of suitable surfactants include Tween20,Tween80; a polyethylene glycol or a polyoxyethylene polyoxypropyleneglycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10%(w/v).

The salt or buffering agent may be any salt or buffering agent, such asfor example, sodium chloride, or sodium/potassium phosphate,respectively. Preferably, the buffering agent maintains the pH of thepharmaceutical composition in the range of about 5.5 to about 7.5. Thesalt and/or buffering agent is also useful to maintain the osmolality ata level suitable for administration to a human or an animal. Preferablythe salt or buffering agent is present at a roughly isotonicconcentration of about 150 mM to about 300 mM.

The formulations of the carrier complex useful in the methods of thepresent invention may additionally contain one or more conventionaladditives. Some examples of such additives include a solubilizer suchas, for example, glycerol; an antioxidant such as for example,benzalkonium chloride (a mixture of quaternary ammonium compounds, knownas “quats”), benzyl alcohol, chloretone or chlorobutanol; anaestheticagent such as for example a morphine derivative; or an isotonic agentetc., such as described above. As a further precaution against oxidationor other spoilage, the pharmaceutical compositions may be stored undernitrogen gas in vials sealed with impermeable stoppers.

The mammal can be any mammal, including, for example, farm animals, suchas sheep, pigs, cows, and horses; pet animals, such as dogs and cats;laboratory animals, such as rats, mice and rabbits. In a preferredembodiment, the mammal is a human.

Utility

Due to the ability of the carrier complexes to cross cell membranes inan energy-independent mechanism, numerous in vivo and in vitroapplications are possible.

The carrier complexes can, for example, be used in vitro, as a researchtool. For example, the carrier complexes can deliver molecules, such asproteins, into a cell so that the functional role of the molecule can bestudied. Such molecules include, for example, cell signaling proteins(e.g., nuclear factor NF-.kappa.B, kinases, such as JAK).

Another in vitro application includes, for example, the delivery of amarker, such as β-galactosidase, into a cell, such as a stem cell,hemopoietic cell, or embryonic cell, to determine progeny (lineage) of acell.

Other in vitro applications include, for example, the delivery of adetectable antibody into a cell to determine the presence of aparticular protein in the cell.

The carrier complexes also have therapeutic uses in vivo. For example,the aromatic cationic peptides can be used for delivering antisensepolynucleotides into a cell of a mammal to down-regulate overexpressionof a protein. Further, the aromatic cationic peptides can be used fordelivering oligonucleotides for RNA interference (RNAi).

RNAi as used herein refers to a cellular mechanism to regulate theexpression of genes or the replication of viruses or bacteria. Themechanism includes the introduction of double stranded RNA (e.g., siRNA)to target a gene's product (typically RNA).

The blood-brain barrier is particularly selective. Thus, another in vivoapplication include delivering molecules across the blood-brain barrier.Such molecule can include, for example, an antibody to β-amyloid in thetreatment of patients with Alzheimers disease.

Typical problems associated with chemotherapeutic agents is achievingadequate levels inside a cell. For example, the chemotherapeutic agentmay be too large or the agent may not be aromatic enough to cross thecell membrane. Thus, another in vivo application includes deliveringchemotherapeutic agents, such as the cytotoxic agents described above,into a cell.

EXAMPLES Example 1 Materials and Methods

Drugs and Chemicals.

[Dmt¹]DALDA and [³H][Dmt¹]DALDA (47 Ci/mmol) were synthesized accordingto methods described previously (Schiller et al., Eur. J. Med. Chem.2000, 35: 895-901; Zhao et al., J. Pharmacol. Exp. Ther. 2002, 302:188-196). [¹⁴C]Gly-Sar (56.7 mCi/mmol) and[³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (50 Ci/mmol) were purchasedfrom Amersham Biosciences (Piscataway, N.J.). All other drugs andchemicals were obtained from Sigma-Aldrich (St. Louis, Mo.).

Cell Culture.

All cell lines were obtained from American Type Culture Collection(Manassas, Va.), and cell culture supplies were obtained from Invitrogen(Carlsbad, Calif.). Caco-2 cells were grown in MEM, whereas SH-SY5Y,HEK293 and Huh7 cells were grown in Dulbecco's modified Eagle's medium.Growing media were supplemented with 10% fetal bovine serum, 200 μg/mlpenicillin, and 100 μg/ml streptomycin sulfate. CRFK cells were grown inMEM+10% horse serum, nonessential amino acids, andpenicillin/streptomycin. All cell lines were maintained at 37° C. in ahumidified atmosphere of 95% air and 5% CO₂.

Assay for Peptide Uptake.

Peptide internalization was studied primarily using Caco-2 cells andsubsequently confirmed with SH-SY5Y, HEK293, and CRFK cells. Monolayersof cells were grown on 12-well plates (5×10⁵ cells/well) coated withcollagen for 3 days. On day 4, cells were washed twice with prewarmedHBSS, and then incubated with 0.2 ml of HBSS containing either 250 nM[³H][Dmt¹]DALDA or 50 μM [¹⁴C]Gly-Sar at 37° C. for various times up to1 h. In a separate experiment, cells were incubated with the sameconcentration of [³H][Dmt¹]DALDA in the presence of unlabeled[Dmt¹]DALDA (1 μM-3 mM) for 1 h at 37° C. For uptake studies at 4° C.,cells were put on ice for 20 min before incubation with [³H][Dmt¹]DALDAor [¹⁴C]Gly-Sar. At the end of the incubation period, cells were washedfour times with HBSS, and 0.2 ml of 0.1 N NaOH with 1% SDS was added toeach well. The cell contents were then transferred to scintillationvials and radioactivity was counted. An aliquot of cell lysate was usedfor determination of protein content using the method of Bradford(Bio-Rad, Hercules, Calif.). To distinguish between internalizedradioactivity from surface-associated radioactivity, an acid-wash stepwas included. Before cell lysis, cells were incubated with 0.2 ml of 0.2M acetic acid/0.05 M NaCl for 5 min on ice.

Assay for Peptide Efflux from CaCo-2 Cells.

Monolayers of Caco-2 cells were grown on 12-well plates (5×10⁵cells/well) for 3 days. On day 4, cells were preloaded with[³H][Dmt¹]DALDA or [¹⁴C]Gly-Sar for 1 h at 37° C. Cells were then washedfour times with 1 ml of ice-cold incubation solution to terminate uptakeand then incubated with 0.5 ml of MEM for 1 h at either 37 or 4° C. tomeasure the efflux of peptide from cells to the incubation medium. Theamount of radioactivity was determined in cell lysates and in theincubation medium. To examine the role of P-glycoprotein on peptideuptake and efflux from cells, [Dmt¹]DALDA uptake and efflux were alsodetermined in the presence of 100 μM verapamil (P-glycoproteininhibitor).

Assay, for Peptide Translocation Across Caco-2 Monolayers.

Monolayers of Caco-2 cells were prepared as described previously (Irieet al., J. Pharmacol. Exp. Ther. 2001, 298: 711-717). Caco-2 cells(2×10⁵) were seeded on microporous membrane filters (24 mm, 0.4 μM)inside Transwell cell culture chambers (Corning Glassworks, Corning,N.Y.). Each Transwell chamber was filled with 1.5 ml of medium in theapical compartment and 2.5 ml in the basolateral compartment. The cellmonolayers were given fresh medium every 1 to 2 days and were used onday 28 for transport experiments. Apical-to-basolateral transport ofpeptides was determined by adding 0.2 μM [³H][Dmt¹]DALDA or 100 μM[¹⁴C]Gly-Sar to the apical compartment, and 50-μl aliquots were removedfrom both apical and basolateral compartments at various times afterpeptide addition for determination of radioactivity counts.

The apparent permeability coefficient was calculated according to thefollowing equation: P_(app)=X/(t·A·Co), where X/t is the rate of uptakein the receiver compartment, A is the diffusion area (4.72 cm²), and Cois the initial concentration in the donor compartment.

Confocal Laser Scanning Microscopy. The uptake of aromatic-cationicpeptides into cells was confirmed by confocal laser scanning microscopy(CLSM) using two fluorescent peptides, [Dmt¹,dnsDap⁴]DALDA(Dmt-D-Arg-Phe-dnsDap-NH₂, where dnsDap isβ-dansyl-1-α-β-diamino-propionic acid) and [Dmt¹,atnDap⁴]DALDA(Dmt-D-Arg-Phe-atnDap-NH₂, where atn isβ-anthraniloyl-L-α-(β-diaminopropionic acid). Caco-2 cells or SH-SY5Ycells were grown as described above and were plated on (35-mm) glassbottom dishes (MatTek, Ashland, Mass.) for 2 days. The medium was thenremoved, and cells were incubated with 1 ml of HBSS containing 0.1 μM ofthe fluorescent peptide at either 4° C. or 37° C. for 15 min. Cells werethen washed three times with ice-cold HBSS and covered with 200 μl ofPBS, and microscopy was performed within 10 min at room temperatureusing a confocal laser scanning microscope with a C-Apochromat 63x/1.2 Wcorr objective (Nikon, Tokyo, Japan). Excitation/emission wavelengthswere set at 340/520 nm for [Dmt¹,dnsDap⁴]DALDA and 320/420 nm for[Dmt¹,atnDap⁴]-DALDA, respectively. For optical sectioning inz-direction, 5 to 10 frames with 2.0 μM were made.

Radioligand Binding Assay Using Cell Membranes.

Specific binding of [³H][Dmt¹]DALDA to cell surface receptors wasdetermined using membranes prepared from Caco-2 and SH-SY5Y cells. After4 days of culture, cells were washed two times with PBS buffer and thenscraped off. Cells were centrifuged at 500 g for 5 min and the pelletstored at −80° C. Cells were homogenized in ice-cold 50 mM Tris-HClbuffer (5 μg/ml leupeptin, 2 μg/ml chymostatin, 10 μg/ml bestatin, and 1mM EGTA, pH 7.4). The homogenate was centrifuged at 36,000 g for 20 min.The pellets were resuspended with 50 mM Tris-HCl buffer. Aliquots ofmembrane homogenates (˜140 μg of protein) were incubated with[³H][Dmt¹]DALDA (15-960 pM) for 60 min at 25° C. Nonspecific binding wasassessed by inclusion of 1 unlabeled [Dmt¹]DALDA. Free radioligand wasseparated from bound radioligand by rapid filtration through GF/Bfilters (Whatman, Maidstone, UK) with a cell harvester (Brandel Inc.,Gaithersburg, Md.). Filters were washed three times with 10 ml of Trisbuffer, and radioactivity was determined by liquid scintillationcounting. Binding affinities (K_(d)) and receptor number (B_(max)) weredetermined using nonlinear regression (GraphPad Software, San Diego,Calif.).

Conjugation of Protein to [Dmt1] DALDA.

[Dmt¹]DALDA was cross-linked to (β-galactosidase (recombinant E. coli,Sigma-Aldrich) using a cross-linker SMCC (succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate)(Pierce). SMCC reactswith amine-containing molecules (Lys⁴ of [Dmt¹]DALDA) to form stableamide bonds. Its maleimide end can then be conjugated to asulfhydryl-containing compound to create a thioether linkage(Bioconjugate Techniques by Greg T. Hermanson, Academic Press, page234-237). β-Gal contains abundant free sulfhydryl groups in its nativestate. The uptake of (β-Gal provides a convenient read-out with the useof X-gal. Briefly, 1 ml of 5×10⁻³M [Dmt¹]DALDA was mixed with 1 mg SMCCin phosphate buffer for 1 h at room temperature. This should result in“activated peptide.” The “activated peptide” was diluted 1:10 withphosphate buffer. 1 mg of β-Gal was added to 1 ml of the 1:10 “activatedpeptide” and mixed at 4° C. for either 2 h or overnight.

Coupling of [Dmt1]DALDA to Cross-Linker SMCC and Confirmation by MassSpectrometry.

SMCC (1 μg) and [Dmt¹]DALDA (5 μg) were dissolved together in 2 ml ofPBS, incubated at room temperature for 30 min, and stored at 4° C. Analiquot of the sample was mixed with matrix (saturated 3-hydroxypicolinic acid (HPA) in 50% acetonitrile, 10 mg/ml ammonium citrate) ina 1:10 ratio, and spotted on a stainless steel target plate. Sampleswere analyzed by Matrix Assisted Laser DesorptionIonization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) (AppliedBiosystems (Voyager DE Pro)) in the positive Reflectron mode.

Conjugation of RNA to [Dmt1]DALDA and Confirmation by GelElectrophoresis.

Synthetic RNA oligo (40 nucleotides long) was phosphorylated at the 5′end using γ-³²P-ATP and polynucleotide kinase. The product was purifiedby gel electrophoresis. 500,000 cpm of gel-purified RNA oligo wasconjugated with [Dmt¹]DALDA in the presence of 1 mg EDC(N-[3-dimethylaminopropyl-N′-ethylcarboiimide]). The product of theconjugation reaction ([Dmt¹]DALDA-RNA oligo) and RNA oligo alone wereanalyzed on 15% polyacrylamide urea gel.

Conjugation of DNA to [Dmt1]DALDA and Confirmation by Mass Spectrometry.

SMCC (1 μg) and [Dmt¹]DALDA (5 μg) were dissolved together in 2 ml ofPBS, incubated at room temperature for 30 min, and mixed withdeprotected 3′-thiol DNA oligo at 4° C. for 24 hours. After incubation,an aliquot of sample was mixed with matrix (saturated 3-hydroxypicolinic acid (HPA) in 50% acetonitrile, 10 mg/ml ammonium citrate) ina 1:10 ratio, and spotted on a stainless steel target plate. Sampleswere analyzed by MALDI-TOF MS.

Carrier complex formation by physical mixing of RNA and [Dmt¹]DALDA-SMCCconjugate. The [Dmt¹]DALDA-SMCC conjugate was prepared as describedabove. The RNA molecules were mixed with the [Dmt¹]DALDA-SMCC conjugatein PBS for 15 min at room temperature before use in cellular uptakestudies.

Carrier Complex Formation by Physical Mixing of Protein and[Dmt1]DALDA-SMCC Conjugate.

The [Dmt¹]DALDA-SMCC conjugate was prepared as described above. Theprotein molecules (i.e. green fluorescent protein, GFP) were mixed withthe [Dmt¹]DALDA-SMCC conjugate for 15 min at room temperature before usein cellular uptake studies.

Assay for [Dmt1] DALDA-RNA Conjugate Uptake into Cells.

Synthetic RNA oligos were phosphorylated at the 5′ end using γ-³²P-ATPand polynucleotide kinase, and the products were purified by gelelectrophoresis. 500,000 cpm of gel-purified RNA oligo was conjugatedwith [Dmt¹]DALDA in the presence of 1 mgN-(3-dimethylaminopropyl-N′-ethylcarboiimide, EDC). Caco-2 cells (1×10⁶)were washed three times in DMEM medium and pre-incubated in DMEM for 5minutes. Cells were then incubated with [Dmt¹]DALDA-[³²P]RNA oligoconjugate or unconjugated RNA (approximately 20,000 cpm) for 60 minutesat 37° C. After incubation, the cells were washed three times in DMEM,incubated with lysis buffer, and radioactivity determined in the celllysate.

Assay for Uptake of RNA Uptake into Cells when Mixed with[Dmt1]DALDA-Cross-Linker Conjugate.

Huh7 cells (1×10⁶ cells/well) were washed with DMEM and then incubatedwith 1.0 ml DMEM containing [³²P]RNA oligo alone or with 40 μl[Dmt¹]DALDA-SMCC conjugate, for 60 min at 37° C. or 4° C. Cells werethen washed four times in DMEM and one time in sodium acetate solutionto reduce nonspecific binding before incubated in lysis buffer for 30min and radioactivity determined in the cell lysate.

Assay for [Dmt1]DALDA-Protein Conjugate Uptake into Cells.

Cells (N₂A neuroblastoma cells or Caco-2) were plated in 96-well plates(2×10⁴ cells/well) and incubated with [Dmt¹]DALDA cross-linked β-Gal orβ-Gal alone for 1 h at 37° C. Cells were then washed 4 times with PBS.The cells were then stained with β-gal staining set (Roche) for at least2 h at 37° C. and examined under the microscope.

Assay for Protein Uptake into Cells when Co-Incubated with[Dmt1]DALDA-SMCC Conjugate.

Huh7 cells (1×10⁴ cells/well) were washed with DMEM and then incubatedwith 0.5 ml DMEM containing 3 μg green fluorescent protein (GFP) alone(A), 3 μg GFP and 40 μl [Dmt¹]DALDA (B), or 3 μg GFP and 40 μl[Dmt¹]DALDA conjugated to SMCC(C) for 60 min at 37° C. 2 ml of cellmedium was then added to cells which were incubated for an additional 24hours in the cell culture incubator. After incubation, cells were washedfour times in cell medium and GFP retained in living cells wasvisualized by confocal laser scanning microscopy. Excitation wasperformed at 340 nm and emission was measured at 520 nm.

Assay for Apoptosis.

Apoptosis was determined with the use of Hoechst dye (Molecular Probes,Eugene, Oreg.) for staining apoptotic nuclei. The Hoechst dye was loadedto cell cultures and incubated for 15 min. Excessive Hoechst dye wasremoved by washing cells with cell medium (free of pH indicator) thecells examined using fluorescent microscopy (excitation at 350 nm andemission at 461 nm).

Example 2 Time Course of Uptake of [Dmt¹]DALDA and Gly-Sar into Caco-2Cells

When incubated with Caco-2 cells at 37° C., [³H][Dmt¹]DALDA was observedin cell lysate as early as 5 min, and steady-state levels were achievedby 30 min (FIG. 1A). The total amount of [³H][Dmt¹]DALDA recovered inthe cell lysate after 1-h incubation represented about 1% of the totaldrug. In contrast, under the same experimental conditions, [¹⁴C]Gly-Sarcontinued to increase from 5 to 45 min (FIG. 1B). The measuredradioactivity is believed to reflect [Dmt¹]DALDA levels, because we havepreviously demonstrated that [Dmt¹]DALDA is resistant against peptidasedegradation (Szeto et al., J. Pharmacol. Exp. Ther., 2001, 298: 57-61).To determine whether the measured radioactivity was associated with cellmembranes, cells were subjected to acid-wash to remove surface binding.FIG. 1C shows that 80.8% of [³H][Dmt¹]DALDA was resistant to acid-washand therefore presumed to be inside the cell. The uptake of [Dmt¹]DALDAwas found to be concentration-dependent over a wide range ofconcentrations (FIG. 1D).

Example 3 Temperature Dependence and Effects of pH on Uptake of[Dmt¹]DALDA and Gly-Sar

When the incubation was carried out at 4° C., the uptake of[³H][Dmt¹]DALDA was slower compared with 37° C., but reached 76.5% by 45min (FIG. 1A) and 86.3% by 1 h (FIG. 1A). In contrast, the uptake of[¹⁴C]Gly-Sar was completely abolished at 4° C. (FIG. 1B). The uptake ofGly-Sar by PEPT1 is known to be pH-dependent, with optimal uptakeoccurring at pH 6.0 (Terada et al., 1999, Am. J. Physiol. 276:G1435-G1441). This was confirmed in our study (FIG. 2B). In contrast,the uptake of [³H][Dmt¹]DALDA was unchanged when pH varied from 4.0 to7.4 (FIG. 2A). The lack of temperature and pH dependence suggests thatthe uptake of [Dmt¹]DALDA in Caco-2 cells is not mediated via PEPT1(peptide transporter 1).

Example 4 Effect of DEPC on [Dmt¹]DALDA and Gly-Sar Uptake

To further demonstrate that PEPT1 is not involved in the transport of[Dmt¹]DALDA, we examined the effect of DEPC (diethylpyrocarbonate; 0.2mM) on [³H][Dmt¹]DALDA and [¹⁴C]Gly-Sar uptake. DEPC is a histidineresidue-modifier reagent that has been shown to inhibit PEPT1 in Caco-2cells (Terada et al., FEBS. Lett., 1996, 394: 196-200). The addition ofDEPC to the incubation medium significantly inhibited [¹⁴C]Gly-Saruptake (FIG. 2D). Surprisingly, DEPC not only did not inhibit[³H][Dmt¹]DALDA uptake but also it actually increased [Dmt¹]DALDA uptakeby 34-fold (FIG. 2C).

Example 5 [Dmt¹]DALDA Internalization in Different Cell Types

To demonstrate that the internalization of [Dmt¹]DALDA was not limitedto Caco-2 cells, we compared the internalization of [Dmt¹]DALDA inseveral different cell lines. An acid-wash step was included todistinguish internalized radioactivity (acid-resistant) fromsurface-bound radioactivity (acid-sensitive). FIG. 3A compares thelevels of acid-resistant radioactivity in Caco-2, SH-SY5Y, HEK293, andCRFK cells. The results show that [³H][Dmt¹]DALDA was taken upextensively in all cell types.

Example 6 Radioligand Binding Assays with [³H][Dmt¹]DALDA

To determine whether [Dmt¹]DALDA was internalized via receptor-mediatedmechanisms, we carried out radioligand ([³H][Dmt¹]DALDA) binding assayswith membranes prepared from Caco-2 cells and SH-SY5Y cells. FIG. 3Bshows the specific binding of [³H][Dmt¹]DALDA to SH-SY5Y membranes. Thecalculated K_(d) value was 118 pM (range 87-149) and the B_(max) valuewas estimated to be 96 fmol/mg protein (range 88-104). This iscomparable with the values obtained using recombinant human μ-opioidreceptor expressed on Chinese hamster ovary cells (G.-M. Zhao and H. H.Szeto, unpublished data). No high-affinity specific binding was observedwith Caco-2 membranes (FIG. 3B). It is known that HEK293 cells do nothave opioid receptors (Blake et al., J. Biol. Chem., 1997, 272:782-790).

Example 7 Efflux of [Dmt¹]DALDA and Gly-Sar from Caco-2 Cells

The achievement of steady-state [³H][Dmt¹]DALDA levels in Caco-2 cellsafter <30 min of incubation suggested that the rate of efflux of thepeptide from the cell was equal to the rate of uptake at that time. Toexamine the efflux of Gly-Sar and [Dmt¹]DALDA from the cell, Caco-2cells were preloaded with [¹⁴C]Gly-Sar or [³H][Dmt¹]DALDA and thenreplaced with fresh medium that did not contain peptide. FIG. 4A showsthat 39% of [¹⁴C]Gly-Sar was found in the medium after 1 h at 37° C. Theefflux of [¹⁴C]Gly-Sar was significantly reduced at 4° C. The efflux of[³H][Dmt¹]DALDA from Caco-2 cells was much faster, with 80% of thepeptide effluxed into the medium by 1 h (FIG. 4A). In contrast to theinternalization of [³H][Dmt¹]DALDA (FIG. 1A), temperature had asignificant effect on the efflux of [³H][Dmt¹]DALDA from the cell (FIG.4A). The efflux of [Dmt¹]DALDA was decreased in cells treated with DEPC(FIG. 4B). The reduction in [³H][Dmt¹]DALDA efflux by DEPC is consistentwith the greatly increased uptake of [³H][Dmt¹]DALDA in the presence ofDEPC (FIG. 2C). On the other hand, the efflux of [³H][Dmt¹]DALDA was notaffected by verapamil, an inhibitor of P-glycoprotein (FIG. 4C).Verapamil also had no effect on cellular uptake of [³H][Dmt¹]DALDA (FIG.4D).

The efflux of [Dmt¹]DALDA out of the cell may be beneficial if uponenzymatic cleavage after cellular uptake of the [Dmt¹]DALDA-proteinconjugate, [Dmt¹]DALDA is effluxed out of the cell while the proteincargo remains inside.

Example 8 Transcellular Transport of [Dmt¹]DALDA and Gly-Sar

Caco-2 monolayers grown in Transwells were used to study theapical-to-basolateral transport of [³H][Dmt¹]DALDA and [¹⁴C]Gly-Sar.FIG. 5 illustrates the transport of [¹⁴C]Gly-Sar and [³H][Dmt¹]DALDA inthe basolateral side at various times after loading the peptide in theapical side of the Transwell. The percentage of [³H][Dmt¹]DALDAtranslocated from the apical to the basolateral side in 60 min (10.4%)was comparable with the percentage of [¹⁴C]Gly-Sar transported (11.9%).The apparent permeability coefficient was estimated to be 1.24×10⁻⁵ cm/sfor [Dmt¹]DALDA and 1.26×10⁻⁵ cm/s for Gly-Sar.

Example 9 Visualization of Cellular Uptake of Aromatic-Cationic PeptidesUsing CLSM

To visualize the uptake and mode of cellular internalization ofaromatic-cationic peptides, two fluorescent peptides([Dmt¹,dnsDap⁴]DALDA and [Dmt¹,atnDap⁴]DALDA) were studied by CLSM. FIG.6 shows the internalization of the fluorescent peptide into Caco-2 cellsafter incubation with 0.1 μM [Dmt¹,dnsDap⁴]DALDA for 15 min at 37° C.The fluorescence appeared diffuse throughout the cytoplasm with noapparent vesicular distribution, suggesting that the uptake of thepeptide did not involve endocytosis and the peptide is not enclosed inan endosome. Note also that the peptide was completely excluded from thenucleus. The internalization of [Dmt¹,atnDap⁴]DALDA into SH-SY5Y cellsafter incubation with 0.1 μM [Dmt¹,atnDap⁴]DALDA for 30 min at 4° C.clearly support a energy-independent non-endocytotic uptake mechanism,because endocytosis is an energy-dependent process.

Example 10 Coupling of Peptides to Cross-Linker SMCC and Confirmation byMass Spectrometry

SMCC (1 μg) and 5 μg of [Dmt¹]DALDA, [Phe¹]DALDA, or[d-Arg-Dmt-Lys-Phe-NH₂] were dissolved together in 2 ml of PBS,incubated at room temperature for 30 min, and stored at 4° C. An aliquotof the sample was mixed with matrix (saturated 3-hydroxy picolinic acid(HPA) in 50% acetonitrile, 10 mg/ml ammonium citrate) in a 1:10 ratio,and spotted on a stainless steel target plate. Samples were analyzed byMatrix Assisted Laser Desorption Ionization-Time-of-Flight MassSpectrometry (MALDI-TOF MS) (Applied Biosystems (Voyager DE Pro)) in thepositive Reflectron mode. The molecular weights of the peptides andtheir respective peptide-SMCC conjugates are indicated on the massspectra (FIG. 7).

Example 11 Peptide Conjugated to a Protein Carlo Brings the ProteinCargo into Cells

Various peptides were cross-linked to β-galactosidase (recombinant E.coli, Sigma-Aldrich) using a cross-linker SMCC (Pierce). SMCC reactswith amine-containing molecules (Lys⁴ of [Dmt¹]DALDA) to form stableamide bonds. The formation of peptide-SMCC conjugates is confirmed bymass spectrometry (FIG. 7). Its maleimide end can then be conjugated toa sulfhydryl-containing compound to create a thioether linkage(Bioconjugate Techniques by Greg T. Hermanson, Academic Press, page234-237). β-Gal contains abundant free sulfhydryl groups in its nativestate. The uptake of β-Gal provides a convenient read-out with the useof X-gal.

Briefly, 1 ml of 5×10⁻³M [Dmt¹]DALDA, [Phe¹]DALDA or[d-Arg-Dmt-Lys-Phe-NH₂] was mixed with 1 mg SMCC in phosphate buffer for1 h at room temperature. This should result in “activated peptide.” The“activated peptide” was diluted 1:10 with phosphate buffer. 1 mg ofβ-Gal was added to 1 ml of the 1:10 “activated peptide” and mixed at 4°C. for either 2 h or overnight.

Cells (N₂A neuroblastoma cells or Caco-2) were plated in 96-well plates(2×10⁴ cells/well) and incubated with β-Gal or β-Gal cross-linked with[Dmt¹]DALDA, [Phe¹]DALDA or [d-Arg-Dmt-Lys-Phe-NH₂] for 1 h at 37° C.Cells were then washed 4 times with phosphate buffer. The cells werethen stained with β-gal staining set (Roche) for at least 2 h at 37° C.and examined under the microscope.

No uptake of β-Gal was observed when Caco-2 cells were incubated withβ-Gal (FIG. 8A). Presence of blue cells indicate uptake of β-Galconjugated with [Dmt¹]DALDA in Caco-2 cells (FIG. 8B). Enhanced uptakeof β-Gal was also found when it was conjugated with[d-Arg-Dmt-Lys-Phe-NH₂] (FIG. 8C) or [Phe¹]DALDA (FIG. 8D). Conjugationof β-Gal with SMCC alone did not enhance uptake.

Similar results were obtained when neuronal N₂A cells or CHO cells(Chinese hamster ovarian cells) were used.

Example 12 Co-Incubation with [Dmt¹]DALDA-SMCC Conjugate Enhances Uptakeof Green Fluorescent Protein (GFP) into Huh7 Cells

Huh7 cells (1×10⁶ cells/well) were washed with DMEM and then incubatedwith 0.5 ml DMEM containing 3 μg GFP alone, 3 μg GFP and 40 μl[Dmt¹]DALDA, or 3 μg GFP and 40 μl [Dmt¹]DALDA conjugated to SMCC for 60min at 37° C. 2 ml of cell medium was then added to cells and incubatedfor an additional 24 hours in cell culture incubator. After incubation,cells were washed four times in cell medium and GFP retained in livingcells was visualized by confocal laser scanning microscopy. Excitationwas performed at 340 nm and emission was measured at 520 μM.

FIG. 9 (top panel) represents images of GFP through 0.8 μM thick centralhorizontal optical section of Huh7 cells. FIG. 9 (bottom panel)represents differential interface contrast images in same field.

Co-incubation of GFP with [Dmt¹]DALDA showed moderately increased greenfluorescence within the cell cytoplasm (FIG. 9B) compared to incubationwith GFP alone (FIG. 9A). No green fluorescence was observed in thenucleus. Co-incubation of GFP with [Dmt¹]DALDA-SMCC conjugate showedeven greater uptake of GFP (FIG. 9C). These data show that [Dmt¹]DALDAcan promote protein uptake into cells by just physical mixing of themodified peptide with the protein, and that chemical conjugation betweenthe peptide and the protein is not necessary.

Example 13 Conjugation of [Dmt]¹ DALDA with an RNA Oligo

Synthetic RNA oligo (40 nucleotides long) was phosphorylated at the 5′end using γ-³²P-ATP in the reaction with polynucleotide kinase. Theproduct was gel-purified for reaction. 500,000 counts per minute ofgel-purified RNA oligo was conjugated in the reaction with 10 mM [Dmt]¹DALDA in the presence of 1 mg EDC(N-[3-dimethylaminopropyl-N′-ethylcarboiimide]). The product ofconjugation reaction ([Dmt]′DALDA-RNA oligo) and control RNA oligo alonewere analyzed on 15% polyacrylamide urea gel. Two distinct bands on thegel indicate the RNA oligo alone and the [Dmt¹]DALDA-RNA oligo conjugate(FIG. 10).

Example 14 Uptake of [Dmt]¹DALDA-RNA Oligo Conjugate into Caco-2 Cells

Caco-2 cells (1×10⁶) were washed three times in DMEM medium andpreincubated in DMEM for 5 minutes before addition of oligos. Then,either [Dmt]¹DALDA-RNA oligo conjugate or unconjugated RNA(approximately 20,000 counts per minute each) were added to the cellmedium and incubated for 60 min at 37° C.

After the incubation, reaction medium was removed and cells washed fourtimes with DMEM and one time in sodium acetate solution to reducenonspecific binding. Finally, the cells were incubated in lysis bufferfor 30 minutes and radioactivity in the cell lysate was measured.

Caco-2 cells exhibited over three times greater uptake of[Dmt¹]DALDA-RNA oligo conjugate as compare to unconjugated RNA oligoalone (FIG. 11). Therefore, [Dmt]¹DALDA promotes passage of RNA oligoacross the cell membrane.

Example 15 Mixing of RNA with [Dmt¹]DALDA-SMCC Linker Increases RNAUptake Into Cells

The carrier complex was formed by physical mixing of RNA and[Dmt¹]DALDA-SMCC conjugate. The [Dmt¹]DALDA-SMCC conjugate was preparedby mixing [Dmt¹]DALDA with the cross-linker SMCC as described underMethods. A single strand 11-mer [³²P]RNA oligo was mixed with the[Dmt¹]DALDA-SMCC conjugate for 15 min at room temperature before use incellular uptake studies.

Huh7 cells (1×10⁶ cells/well) were washed with DMEM and then incubatedwith 1.0 ml DMEM containing the [³²P]RNA oligo (100,000 cpm) alone orwith 40 ml [Dmt¹]DALDA-SMCC conjugate at 37° C. or 4° C. Cells were thenwashed four times in DMEM and one time in sodium acetate solution toremove nonspecific binding before incubated in lysis buffer for 30 minand retained radioactivity determined.

Co-incubation of RNA oligo with [Dmt¹]DALDA-SMCC at 37° C. increaseduptake of the RNA oligo as a function of time (FIG. 12A). At one hour,the uptake of RNA oligo in the presence of [Dmt¹]DALDA-SMCC wasincreased ˜20-fold compared to incubation with RNA alone. The uptake ofRNA was significantly enhanced by [Dmt¹]DALDA-SMCC even at 4° C. (FIG.12B). These data show that it is possible to enhance RNA uptake withoutchemical conjugation with [Dmt¹]DALDA. The uptake at 4° C. indicatesuptake by energy-independent non-endocytotic processes, consistent withthe ability of [Dmt¹]DALDA to penetrate cell membranes by passivediffusion.

In addition to [Dmt¹]DALDA-SMCC, co-incubation with [Phe¹]DALDA-SMCC or[d-Arg-Dmt-Lys-Phe-NH₂]-SMCC also enhanced the uptake of the 11-mer RNAoligo. FIG. 12C shows the increase in RNA uptake when incubated with thethree different peptide-SMCC conjugates for 15 min at 37° C.

Co-incubation with [Dmt¹]DALDA-SMCC conjugate can also promote thecellular uptake of a much larger RNA molecule (1350-mer) as shown inFIG. 13, although not as much as for a smaller oligo.

Example 16 Conjugation of [Dmt¹]DALDA with a DNA Olio

SMCC (1 μg) and [Dmt¹]DALDA (SS002; 5 μg) were dissolved together in 2ml of PBS, incubated at room temperature for 30 min, and mixed withdeprotected 3′-thiol DNA oligo at 4° C. for 24 hours. After incubation,an aliquot of sample was mixed with matrix (saturated 3-hydroxypicolinic acid (HPA) in 50% acetonitrile, 10 mg/ml ammonium citrate) ina 1:10 ratio, and spotted on a stainless steel target plate.

The formation of the DNA-[Dmt¹]DALDA conjugate was confirmed byMALDI-TOF MS. The molecular weights of 3′-thiol DNA oligo and[Dmt¹]DALDA-DNA covalent complex were found to be 6392 and 7171,respectively (FIG. 14A).

Example 17 Uptake of [Dmt]¹DALDA-DNA Oligo Conjugate into Caco-2 Cells

A 3′-thiol-modified 20-mer DNA was conjugated to [Dmt¹]DALDA using SMCC,and the formation of the conjugate was confirmed by mass spectroscopy.Both conjugated and unconjugated DNA oligos were radiolabeled at the5′-end with ³²P and gel-purified (FIG. 14B).

Neuronal N₂A (1×10⁶ cells/well) cells were washed with DMEM andincubated with 1 ml DMEM containing either [Dmt¹]DALDA-conjugated orunconjugated DNA oligo (˜100,000 dpm) for 2 h or 19 h at 37° C. and 5%CO₂. Cells were then washed four times in DMEM and one time in sodiumacetate solution to reduce nonspecific binding. The cells were thenincubated in lysis buffer for 30 min and retained radioactivitydetermined.

Uptake of DNA conjugated with [Dmt¹]DALDA was greater compared tounconjugated DNA after 19 h of incubation (FIG. 15), indicating that DNAuptake can be enhanced by conjugation to [Dmt¹]DALDA.

Example 18 Peptides and Peptide-SMCC Conjugates are not Toxic to Cells

Neither the peptides nor the peptide-SMCC conjugates are toxic to cellsin culture. Treatment with [Dmt¹]DALDA (1 nM to 10 μM) for 24 h had noeffect on cell viability as measured by the MTT assay (MTS assay,Promega, Madison, Wis.) in N₂A cells (FIG. 16), SH-SY5Y cells or Caco-2cells. Similar studies with [D-Arg-Dmt-Lys-Phe-NH₂) also showed noeffect on cell viability.

Incubation of cells in culture with the peptide-SMCC conjugates also didnot affect cell viability as measured by the uptake of trypan blue.Trypan blue is only taken by cells with increased membrane permeability.Huh7 cells (1×10⁶) were washed three times in DMEM, and 1 ml of freshmedium, or media containing 50 μl of 1 mM [Dmt¹]DALDA-SMCC conjugate,[D-Arg-Dmt-Lys-Phe-NH₂]-SMCC conjugate, or [Phe¹]DALDA-SMCC conjugate,and incubated at 37° C. for 24 hours at 5% CO₂. Cells were then washedthree times with DMEM, and 1 ml of 0.4% trypan blue was added to thecells for 2 minutes. Excessive dye was removed by washing cells in cellmedium and the cells were examined by light microscopy.

Examination of cells by light microscopy demonstrated that cellsincubated with media alone showed minimal trypan blue uptake. Noincrease in trypan blue uptake was observed in cells incubated with[Dmt¹]DALDA-SMCC, [D-Arg-Dmt-Lys-Phe-NH₂]-SMCC, or [Phe¹]DALDA. Incontrast, incubation of cells with DEPC (dicthylpyrocarbonatc) resultedin significant uptake of trypan blue.

Incubation of cells in culture with [Dmt¹]DALDA-SMCC conjugate also didnot induce apoptosis in Huh7 cells in culture. Huh7 cells (1×10⁶cells/well) were washed three times in DMEM, and 1 ml of fresh mediumwas applied. Then, either 50 μl of modified [Dmt¹]DALDA (1 mM) in PBS orPBS only (control) were added to the cell medium and incubated at 37° C.for 24 hours at 5% CO₂. After the incubation, 1 ml of Hoechst dye(Molecular Probes, Eugene, Oreg.) for staining apoptotic nuclei wereadded to cells and incubated for additional 15 min. Excessive Hoechstdye was removed by washing cells with cell medium (free of pH indicator)and cells treated with [Dmt¹]DALDA-SMCC conjugate were compared withcontrol cells using fluorescent microscopy (excitation at 350 nm andemission at 461 μM). Apoptosis is indicated by concentration offluorescence in the nuclei. FIG. 17 demonstrates that the level ofapoptosis in Huh7 cells treated with [Dmt¹]DALDA-SMCC is the same as incontrol cells.

What is claimed is:
 1. A method for delivering a molecule to a cell, themethod comprising contacting the cell with a carrier complex, whereinthe carrier complex comprises the molecule conjugated to an aromaticcationic peptide, wherein the aromatic cationic peptide is selected fromthe group consisting of: Tyr-D-Arg-Phe-Lys-NH₂ (DALDA),2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ (Dmt¹-DALDA), Phe-D-Arg-Phe-Lys-NH₂(Phe¹-DALDA), D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, and2′,6′-Dmp-D-Arg-Phe-Lys-NH₂ (Dmp¹-DALDA); and wherein the molecule is alipid.
 2. A method according to claim 1, wherein the peptide has theformula Tyr-D-Arg-Phe-Lys-NH₂ (DALDA).
 3. A method according to claim 1,wherein the peptide has the formula 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂(Dmt¹-DALDA).
 4. A method according to claim 1, wherein the peptide hasthe formula Phe-D-Arg-Phe-Lys-NH₂ (Phe¹-DALDA).
 5. A method according toclaim 1, wherein the peptide has the formulaD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.
 6. A method according to claim 1, whereinthe peptide has the formula 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂ (Dmp¹-DALDA). 7.A method according to claim 1, wherein the cell is a bacterial cell. 8.A method according to claim 1, wherein the cell is a plant cell.
 9. Amethod according to claim 1, wherein the cell is an animal cell.
 10. Amethod according to claim 9, wherein the animal cell is a mammaliancell.
 11. A method according to claim 9, wherein the cell is a neuronalcell.
 12. A method according to claim 9, wherein the cell is a renalepithelial cell.
 13. A method according to claim 9, wherein the cell isan intestinal epithelial cell.
 14. A method according to claim 9,wherein the cell is a vascular endothelial cell.
 15. A method accordingto claim 14, wherein the endothelial cell is a blood-brain barrierendothelial cell.
 16. A method according to claim 9, wherein the cell isa glial cell.
 17. A method according to claim 9, wherein the cell is ahepatocyte.
 18. A method according to claim 1, wherein thearomatic-cationic peptide comprises a linker.
 19. A method according toclaim 1, wherein the molecule comprises a linker.
 20. A method accordingto claim 1, wherein the molecule and aromatic cationic peptide arechemically bonded.